MiR-15a-5p regulates myocardial fibrosis in atrial fibrillation by targeting Smad7.
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ABSTRACT: Background
At present, there is no effective treatment for myocardial fibrosis in atrial fibrillation (AF). It is reported that miR-15a-5p is abnormally expressed in AF patients but its specific role remains unclear. This study aims to investigate the effect of miR-15a-5p in myocardial fibrosis.Methods
Left atrial appendage (LAA) tissues were collected from AF and non-AF patients. In lipopolysaccharide (LPS) stimulated H9C2 cells, miR-15a-5p mimic, inhibitor, pcDNA3.1-Smad7 and small interfering RNA-Smad7 (siRNA-Smad7) were respectively transfected to up-regulate or down-regulate the intracellular expression levels of miR-15a-5p and Smad7. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to determine the expression levels of miR-15a-5p, Smad7, transforming growth factor β1 (TGF-β1) and collagen I. Cell counting kit-8 (CCK-8) and ethylene deoxyuridine (EdU) were used to determine cell viability and proliferation capacity, respectively. Dual-luciferase was used to detect whether miR-15a-5p interacted with Smad7, hydroxyproline (HYP) and Hematoxylin-Eosin (HE) staining were used to detect tissue fibrosis.Results
The expression levels of miR-15a-5p, TGF-β1 and collagen I were up-regulated, while Smad7 was down-regulated in AF tissues and LPS-stimulated cells. MiR-15a-5p mimic can inhibit the expression of Smad7, and the dual-luciferase experiment confirmed their interaction. MiR-15a-5p inhibitor or pcDNA3.1-Smad7 can inhibit LPS-induced fibrosis and cell proliferation, while siRNA-Smad7 can reverse the changes caused by miR-15a-5p inhibitor.Conclusion
We combined clinical studies with LPS-stimulated H9C2 cell model to validate the role of miR-15a-5p in the regulation of Smad7 and fibrosis. Taken together, the miR-15a-5p/Smad7 pathway might be a potential target for AF therapy.
SUBMITTER: He D
PROVIDER: S-EPMC8697763 | biostudies-literature |
REPOSITORIES: biostudies-literature
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2018-11-22 | PXD006911 | Pride
Project description:Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated ?IIb?3 activation and ?-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of ?IIb?3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced ?IIb?3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcR?-mediated phosphorylation of Syk and PLC?2, GPVI downstream signaling molecules, but effects of miR-15a-5p on ?IIb?3 activation did not extend to other ITAM-signaling receptors (Fc?RIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential "master-miR" for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.
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